Some tumor cells have a nearly absolute requirement for exogeneously supplied asparagine, while others can fulfill their requirements by endogenous biosynthesis. For this reason the enzyme responsible for the breakdown of asparagine has been used as a chemotherapeutic agent. The administration of inhibitors of the asparagine biosynthesis mechanism has also met with some therapeutic success. To enhance the effectiveness of selective tumor cell destruction, a complete understanding of asparagine biosynthesis is necessary. Much, in fact, remains unknown concerning the biosynthesis of asparagine. This ranges from determining the factors that control the asparagine synthesis rate in vivo to describing the metabolic needs for asparagine. We have recently shown that asparagine synthetase from both pancreas and liver can exist in three molecular forms: a 113,000 alpha form, 57,000 beta form and a 26,000 gamma form. A dietary deficiency in asparagine results in the hepatic asparagine synthetase being in the alpha and beta forms. When animals are given dietary asparagine, only beta asparagine synthetase is found. Treatment of animals with asparaginase also results in the appearance of the alpha form of asparagine synthetase. We have shown that the crude beta form can be converted to the alpha form by incubation with glutamine and magnesium. We propose to examine the three different forms of asparagine synthetase and to establish their role in the control of asparagine metabolism in both normal and tumor cells. We have recently found that the alpha-amino nitrogen of asparagine is a major source for glycine nitrogen. The aminotransfer reaction is catalyzed by an asparagine-glyoxylate amino transferase presently being characterized. We also have found that the source of glyoxylate for this asparagine-dependent glycine synthesis is from 4-hydroxy-2-ketoglutarate that may arise from breakdown of hydroxyproline or hydroxylation of glutamate. The relative role of these pathways is being evaluated. Further work has shown that asparagine can be used in glucose production by first transamation to alpha-Ketosuccinamide followed by hydrolysis to oxaloacetate. Evidence for this pathway as the primary route of asparagine breakdown is being accumulated.